Stem cells have the potential to revolutionize medical practice by enabling regenerative medicine. This proposal aims to develop highly efficient generation of human induced pluripotent stem cells (hiPSCs) by utilizing a new class of nanoparticle (NP)-based vectors. These NPs are capable of delivering reprogramming transcription factors (TFs), namely OCT4, SOX2, KLF4 and c-MYC. Current reprogramming systems raise safety concerns because they utilize viral expression of the four key TFs, resulting in hiPSCs with multiple viral integrations. Safer strategies for generating hiPSCs have recently been demonstrated by introducing cell- penetrating peptide-fused reprogramming TFs into human somatic cells, averting any potential dangers of genetic manipulation. Still, effective delivery of TFs remains a key obstacle in creating hiPSCs. Three research groups at UCLA with expertise covering synthetic chemistry, nanoparticles, microfluidics (Tseng and Lu) and stem cell biology (Pyle) have collaborated and accomplished preliminary results consisting of the following fundamental proof-of-concept studies: (i) self-assembly production of supramolecular nanoparticles (SNPs) for delivery of TFs, (ii) (ii) a single protein nano-capsule technology, (iii) digital microreactors for large-scale screening, (iv) microfluidic image cytometry (MIC) technology for quantitative phenotyping of single hiPSCs, and (v) extensive experience in generating hiPSCs. The proposed research will leverage the multidisciplinary team to implement the following two specific aims: 1) We will synthesize a variety of polymer building blocks, cross linkers, functional ligands, as well as, nano- capsules containing the four reprogramming TFs for self-assembly of SNP-based delivery vectors. 2) We will generate a combinatorial library of reprogramming TF-encapsulated SNPs (i.e., OCT4/SOX2/ KLF4/c-MYC?SNPs) by performing ratiometric mixing of the four TF-containing nano-capsules, polymer building blocks, cross linkers and functional ligands. Human ESC-derived fibroblast cells with an OCT4-EGFP reporter will be employed as target cells. Subsequently, the MIC technology will be employed to quantify reprogramming performance by measuring pluripotent markers and colony formation in the SNPs-treated cells. If the efficiency is too low, we propose to co-deliver apoptotic inhibitors (e.g., siRNA_p53) with the four reprogramming TFs using siRNA/TFs?SNPs-based vectors. We anticipate that combination of apoptotic inhibitors and reprogramming TFs could dramatically increase the efficiency of reprogramming. PUBLIC HEALTH RELEVANCE: The long-term objective of this research proposal is to explore the use of supramolecular nanoparticles (SNPs) as a new category of artificial vector for simultaneous delivery of the four reprogramming transcription factors (i.e., OCT4, SOX2, KLF4 and c-MYC) into human somatic cells to induce reprogramming to the induced pluripotent cell (iPSC) state.